Circadian (daily) rhythms are a crucial component of human health that regulates sleep, alertness, hormones, metabolism, and many other biological processes. The ultimate explanation for the mechanism of circadian oscillators will require characterizing the structures, functions, and interactions of the molecular components of these clocks. The current project is to elucidate the basic principles of circadian clocks at a biophysical/molecular level in a model system, the prokaryotic cyanobacteria, where genetic/biochemical studies have identified three key clock proteins, KaiA, KaiB, and KaiC. These three proteins can reconstitute a circadian oscillator in vitro; this remarkable demonstration has led to a re-evaluation of our understanding of circadian clocks in all organisms, including mammals. Moreover, the crystal structures of the KaiA, KaiB, and KaiC proteins have been reported-these are the first clock proteins to have their 3-D structures determined. The advent of atomic resolution structures of the molecular components of this circadian pacemaker marks a dramatic watershed in circadian research by ushering in truly molecular analyses of circadian mechanisms. The current project will determine the molecular basis of the core clockwork by genetic, biochemical, structural, and phylogenetic approaches. Three critically important unanswered questions in chronobiology are to explain how a biochemical mechanism (i) can be temperature compensated, (ii) keep time so precisely over such a long time constant (~24 h), and (iii) modulate chromosomal topology to confer output rhythms of gene expression. This project will face these issues head-on. Temperature compensation of this biological clock will be investigated by screening for temperature dependent mutants of KaiC, KaiB, and KaiA in vivo. These mutations will be mapped onto the 3-D structures of the proteins to generate specific hypotheses that will be tested by novel in vitro biochemical analyses and targeted mutations. The rate constants and other biochemical data that result from the analyses of these mutants will be integrated with our previous data to generate a mathematical model that accounts for the 24 h time constant of the in vitro oscillator. The hypothesis that KaiC monomer exchange is responsible for the genetic dominance/recessive relationships observed for co-expression studies in vivo will be tested by biophysical/biochemical analyses and visualization of monomer exchange by cryo-electron microscopy. Finally, the linkage between this core clockwork and the global orchestration of gene expression over the daily cycle will be illuminated by isolating the KaiC-containing clock protein complex (chronosome) from intact cells and testing the hypothesis that the biochemical action of KaiC and/or the chronosome is that of a DNA/RNA helicase. PUBLIC HEALTH RELEVANCE: This project will clarify circadian mechanisms at molecular levels that were heretofore unreachable. Biological clocks have been found to be crucial for physical and mental health; cancer, metabolic disorders, cardiovascular disease, and depression are associated with the disruption of these timing systems by genetic and/or environmental disturbance (i.e., by shiftwork or jet-lag). Knowledge of circadian mechanisms along with the development of therapies to properly phase sleep will allow us to enhance health, performance, and well-being in addition to improving the quality of life for depressed subjects.